Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
  • A61L27/42—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having an inorganic matrix
  • A61L27/425—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having an inorganic matrix of phosphorus containing material, e.g. apatite
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/14—Macromolecular materials
  • A61L27/20—Polysaccharides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
  • A61L27/3608—Bone, e.g. demineralised bone matrix [DBM], bone powder
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3641—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the site of application in the body
  • A61L27/3645—Connective tissue
  • A61L27/365—Bones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
  • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
  • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
  • A61L27/3821—Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
  • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
  • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
  • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
  • A61L27/3843—Connective tissue
  • A61L27/3847—Bones

Definitions

• the present invention relates to a composite material for use in the preparation in of bone substitutes or grafts for the regeneration or formation of bone tissue in oncological , orthopaedic and spinal surgery.
• Such a process may be due to various causes:
• diskectomy removal of the disk
• spinal fusion fusion of the two adjacent vertebrae
• Vertebrae can be fused together by a special surgical technique that prevents any movement between them.
• spinal fusion may also require additional fixing at the back of the two vertebrae, using rigid metal instruments of various kinds and sizes, such as screws, plugs, pins, plates, intervertebral connectors in various materials, with or without a screwable thread (for example, titanium), to prevent the vertebrae from slipping on one another with consequent compression and loss of alignment, while fusion is established.
• rigid metal instruments such as screws, plugs, pins, plates, intervertebral connectors in various materials, with or without a screwable thread (for example, titanium), to prevent the vertebrae from slipping on one another with consequent compression and loss of alignment, while fusion is established.
• the material used for bone grafts was of bovine origin, or it was constituted by fragments from the tibia, fibula; femur or iliac crest of autologous or heterologous origin, with a fusion success rate ranging between 63 and 95%.
• the fusion process is similar to that which occurs following bone fracture and is not visible on X-ray till six weeks after surgery.
• the vertebrae can be fused in the intervertebral space and/or to the front between the two adjacent vertebral bodies and/or to the back between adjacent transverse processes, laminae, or between other posterior elements of the vertebrae, according to the pathology that the surgery in question is intended to treat.
• Allogenic bone is sure to have less osteoinductive activities.
• Bone is constituted by cells immersed in an extracellular matrix, 30-35% of the dry weight of which is represented by organic matrix (formed by fibres of collagen and glycosaminoglycans including hyaluronic acid), and inorganic substances (including calcium phosphate, calcium and magnesium fluoride) deposited among the collagen fibrils during the mineralisation phase.
• organic matrix formed by fibres of collagen and glycosaminoglycans including hyaluronic acid
• inorganic substances including calcium phosphate, calcium and magnesium fluoride
• Bone metabolism is regulated by hormones and growth factors mainly released by platelets, macrophages, fibroblasts or other types of cell, and chiefly includes, for example, proteins such as BMP, TGF, PDGF, FGF, EGF, IGF and VEGF that can have both a osteoinductive and angiogenic effect on the mesenchymal cells of bone marrow.
• proteins such as BMP, TGF, PDGF, FGF, EGF, IGF and VEGF that can have both a osteoinductive and angiogenic effect on the mesenchymal cells of bone marrow.
• these polymers can actually be toxic, because they release lactic acid as they degrade, and moreover they may induce an inflammatory response thus inhibiting the bone regeneration process.
• Ceramics too like hydroxyapatite, tribasic calcium phosphate, and calcium sulphate, have been widely used in bone regeneration because they are biocompatible and have osteoinductive potential.
• proteins and other molecules of the extracellular matrix for the formation of porous and/or fibrous structures (such as collagen, laminin, fibronectin, and hyaluronic acid) that enhance osteoblast migration and differentiation because they can be loaded with osteoinductive trophic factors.
• porous and/or fibrous structures such as collagen, laminin, fibronectin, and hyaluronic acid
• the main trophic, osteoinductive factors are BMP and TGF, and they are able to direct stem cells to differentiate into osteoblasts and subsequently osteocytes.
• BMP was first isolated from demineralised bone specimens. Indeed, as early as 1965, it was demonstrated that such demineralised matrices (DM) induced the formation of new bone structures ( Urist MR, Science 150: 893, 1965 ).
• the process of producing DM consists in pulverising bone samples into particles with a diameter of 70-450 ⁇ m prior to partially or totally demineralising them with 0.5 N of HCL.
• This process enables the total or partial maintenance of the organic component of the bone tissue, ensuring also the integrity of the proteins (and therefore of the gowth factors) contained therein.
• scaffolds are autologous and/or allogenic bone grafts, demineralised bone matrices, ceramics, bone substitutes constituted by molecules of extracellular matrix (such as collagen and glycosaminoglycans), even though it is obvious that only bone grafts and DM can be defined as osteoinductive, because of the intrinsic presence of differentiating factors.
• osteogenic and osteo-conductive potential of the scaffolds listed above can be considerably increased by introducing bone-progenitor cells, possibly derived from:
• bone graft for use in spinal orthopaedic, neuro-maxillofacial and dental surgery, in orthopaedic surgery to the shoulder, hand and foot, in oncological surgery and in all those pathologies requiring the regeneration/formation of new bone tissue (hence also in pathologies where the fusion of two adjacent bones is indispensable), such as in the following examples:
• WO93/20858 discloses a composite material paste for bone replacement
• WO95/01181 discloses a composite material which is a monolayer matrix comprising hyaluronic acid derivatives and matrix materials e.g hydropxypatite.
• the Applicant has found a composite material comprising:
• This material may be used in the preparation of a multilayer composite material containing as the inner matrix the aforementioned composite material which is associated with at least one layer comprising a hyaluronic acid derivative.
• the aforementioned composite material and the derived multilayer composite may be used in the preparation of bone substitutes or grafts for the regeneration or formation of bone tissue in surgery.
• the present invention therefore relates to bone grafts or bone substitutes consisting of the aforesaid composite material or the derived multilayer composite material.
• the present invention further relates to a process for preparing the aforementioned multilayer composite material which comprises the following steps:
• the substitutes/grafts of bone tissue according to the present invention are absolutely innovative because they are both osteoconductive and osteoinductive, able therefore to induce the process of cellular osteogenesis.
• the new bone substitutes/grafts have in fact the following properties:
• Orthopaedic surgery to the spine that may require the application of the bone graft that is the subject of the present invention includes:
• All the above-listed types of bone graft/fusion can be performed by introducing the graft between the two adjacent vertebrae or in front of and/or behind them, according to the pathology to be surgically treated, as described earlier.
• the main components present in the composite material or in the inner matrix of the derived multilayer composite materials which form the new bone substitutes/grafts according to the present invention are hyaluronic acid (HA) and/or the derivatives thereof, in association with DM or hydroxyapatite and/or tribasic calcium phosphate salts, or, possibly, with granules and/or powders of autologous and/or allogenic bone and/or bone of animal origin.
• HA hyaluronic acid
• DM or hydroxyapatite and/or tribasic calcium phosphate salts or, possibly, with granules and/or powders of autologous and/or allogenic bone and/or bone of animal origin.
• HA is a hetero-polysaccharide composed of alternate residues of D-glucuronic acid and N-acetyl-D-glucosamine. It is a linear chain polymer with a molecular weight that may vary between 50,000 and 13 x 10 6 Da, according to the source from which it is obtained and the methods used to prepare it It is present in nature in the pericellular gels, in the fundamental substance of the connective tissue of vertebrate organisms (of which it is one of the main components), in the synovial fluid of joints, in the vitreous humor and in the umbilical cord.
• the HA used in the present invention may derive from any source, for example it can be extracted from rooster combs (European patent No. 0138572 B1 ), or it can be obtained by fermentation (European patent application No. 0716688 ), or by technological means (Italian patent application No. PD94A000042 ) and its molecular weight can range between 400 and 3x10 6 Da, in particular between 1x 10 5 Da and 1x10 6 Da, and more particularly between 200,000 and 750,000 Da.
• HA derivatives that can be used in the formation of the bone substitutes/grafts that are the subject of the present invention are listed hereafter:
• the HA derivatives listed above that have proved to be particularly important in forming the new bone grafts are the esters of hyaluronic acid, preferably the benzyl ester (Hyaff ®11), esterified to a percentage of between 50 and 100%, and preferably between 75 and 100%.
• HA main ingredient of the extracellular matrix, modulates many different processes such as proliferation, migration, cell differentiation and angiogenesis through the membrane receptor CD44, and that it also has other functions, such as tissue hydration and joint lubrication.
• HA derivatives in the formation of fibres (European patent No. 0618817 B1 ) which, when made into a non-woven fabric, constitute a three-dimensional matrix that can be used above all in the field of dermatology (in the wound-healing process), thanks to HA's strong capacity for chemotaxis, favouring cell recruitment at the application site.
• non-woven fabrics based on hyaluronic acid derivatives can therefore be defined as osteoinductive in cases where the recruited cells are subsequently destined to differentiate into osteoblasts.
• the aforesaid three-dimensional structures can be loaded with mesenchymal cells and maintained in vitro for as long as is necessary to favour cell proliferation and/or differentiation (European patent No. 0863776 B1 ).
• the new bone substitutes/grafts further subject of the present invention are multilayer, composite structures, mainly constituted by hyaluronic acid and the derivatives thereof in association with hydroxyapatite and/or tribasic calcium phosphate, and/or with DM, and/or with bone granules of autologous, allogenic or animal origin.
• They are called multilayer because they are composed of at least 2 and preferably 3 layers with the inner matrix consisting of the composite material according to the present invention containing the aforementioned components (i) and (ii), variously assembled and processed, sandwiched between them.
• the main component of the above-described layers is a hyaluronic acid derivative (as previously listed), preferably an ester derivative, and more specifically the benzyl ester with a percentage of esterification ranging between 50 and 100%, preferably between 75 and 100%, more preferably in the form as listed below:
• the aforementioned films may also be of another HA derivative, such as those listed above.
• the matrix inside the structure claimed by the present Applicant may be constituted by 2 or more components and may therefore be in the form of a composite association of materials that are listed and described below:
• All the above matrices can then be immersed in polymers of various kinds to make the final matrix more compact and to fix them to the layer/s.
• the polymers chosen for their soaking and fixing qualities are the following:
• thermoplastic polymers such as poly-lactic and poly-glycolic acid or poly-caprolactone acid are used, all the above matrices can be fixed to the previously described layer/s by a process of heat treatment of the outer edges to prevent the "sandwich" from coming apart.
• the matrices may undergo a needle-punching process, together with the layers they are to be sandwiched between (European patent No. 0618817 B1 ). Said process can be performed only on the outer edges of the "sandwich” or on the whole, multilayer structure.
• the needle-punching process is possible when thermoplastic polymers are used too, in which case it can be performed at a high temperature to create fusion points between the different fibres.
• the structures can also be sewn with suture thread based on Hyaff or another biocompatible and bioabsorbable polymer.
• the multilayer, composite structures that are the subject of the present invention can also take the form of three-dimensional, bag-shaped structures intended as fillers for vertebral bodies that have been surgically emptied following infections or cancer, or for use in all types of orthopaedic surgery requiring the formation of new bone tissue for the regeneration of tissue that has been damaged or surgically removed.
• All the composite materials according to the present invention can be loaded with bone marrow cells taken from the patient directly in the operating theatre while undergoing the type of orthopaedic surgery that requires their application, or a few days before the graft is due to be performed, to allow the purification and expansion in vitro of the mesenchymal cells in the marrow, so that these can then be loaded into the structure that is the subject of the present invention, either undifferentiated and/or partially or completely differentiated into osteoblasts/osteocytes.
• said new composite structures can also be loaded with allogenic bone marrow cells, possibly purified, expanded and differentiated in vitro.
• Hyaff 11 in DMSO European patent No. 0216453 B1
• DMSO European patent No. 0216453 B1
• the solution is passed through an extruder with a slit 20 cm long and 200 ⁇ m wide; the extruder is immersed in a coagulating bath constituted by 10 litres of ethanol-water at a ratio of 90:10.
• the solid film that is formed is then passed into two subsequent baths filled with, respectively, ethanol-water at a ratio of 80:20 and ethanol alone.
• the film is dried and cut to size.
• 230 g. of sodium chloride crystals with a granule size of between 200 and 350 ⁇ m is mixed with 6.6 g. of citric acid with a granule size of less than 200 ⁇ m and with 8.5 g. of bicarbonate of soda with a granule size of between 140 and 400 ⁇ m.
• the mixture is then supplemented with 20 g. of resorbable hydroxyapatite in granules sized 200-250 ⁇ m (or more), and/or DM and/or tribasic calcium phosphate and/or calcium sulphate.
• Said mixture of salts is then further supplemented with 60 ml of a solution of Hyaff 11 in DMSO at a concentration of 180 mg/ml, and the components are mixed for at least 1 hour.
• the paste thus obtained is spread into flat shapes measuring, for example 5x15 cm, with a thickness, for example, of between 2 and 5 mm, preferably 3 mm. Said shapes are then sprinkled with a mixture of salts constituted by:
• the product thus obtained is subsequently immersed for at least 1 hour in a solution constituted by water-ethanol at a ratio of 70:30.
• the product is then washed repeatedly in water to eliminate the sodium chloride (NaCl) completely.
• the sponges thus obtained are subsequently freeze-dried.
• the product is immersed in 1 litre of solution constituted by Hyaff 11 esterified to a degree of 50% (Hyaff 11 p50) in water at a concentration of 9 g./l. Subsequently, said solution is de-pressurised with a vacuum pump set at a pressure of less than 800 mbar for at least 0.5 minutes then returned to ambient pressure.
• a vacuum pump set at a pressure of less than 800 mbar for at least 0.5 minutes then returned to ambient pressure.
• This cycle is repeated at least 5 times.
• the material thus obtained is freeze-dried.
• Said solution containing hydroxyapatite and/or bone tissue derivatives is de-pressurised with a vacuum pump set at less than 600 mbar for at least 0.5 minutes.
• the above-described cycle is repeated at least 5 times.
• freeze-drying is performed as follows:
• the freeze-dried product is placed in a glass reactor with a cooling system, to which 150 ml of acetone and 1 g. of CMPI (chloro-methyl-pyridinium iodide) has been added.
• CMPI chloro-methyl-pyridinium iodide
• the product is then dried in a flow of nitrogen for at least 8 hours, and subsequently placed in a vacuum for at least 8 hours.
• a solution is prepared that is constituted by 375 mg of hyaluronic acid in 25 ml of phosphate buffer at pH 7.2.
• the solution is mixed with 8 grams of DM and/or hydroxyapatite and/or other biocompatible and biodegradable ceramics in the form of granules with a granule size of 2mm.
• the matrix thus formed is poured into a container measuring 10 x 10 cm and is ready to be freeze-dried.
• the spongy structure thus obtained is placed between two layers of non-woven fabric constituted by Hyaff11 p80.
• the multilayer structure then undergoes a needle-punching process (as described in European patent No. 0618817 B1 ), to enable and favour the joining of the two layers of non-woven fabric and the matrix between them.
• the surfaces thus prepared are then coated with the material of choice (non-woven fabric, or tissue or film, preferably of Hyaff 11), exerting slight pressure on it.
• the material of choice non-woven fabric, or tissue or film, preferably of Hyaff 11
• the product is then immersed in a bath of ethanol-water 80:20 for 1 hour and then washed repeatedly with pure ethanol.
• the final composite product is washed in water and freeze-dried.
• Hyaff 11 p75 fibre is mixed for at least 10 minutes with 84 cc of a solution constituted by hyaluronic acid in an aqueous solution with a concentration of 18-19 mg/ml.
• Said mixture is supplemented with 20 g. of granules of DM and kneaded for at least 15 minutes.
• the paste thus obtained is subsequently spread into squares measuring, for example, 10x10 cm with a thickness of 2-3 mm.
• the inner matrix thus formed is placed between 2 layers of Hyaff 11, made into a non-woven or woven fabric, aventi uguali dimensioni and the composite multi-layer product obtained is calendered and finally freeze-dried.
• the freeze-dried pieces can be cut to size.
• the pieces can be treated by one or other of the following procedures:
• the pieces treated as described in point 1 and those treated as described in point 2 are both washed twice for at least 1 hour (under mechanical stirring) with 1 litre of a solution formed by ethanol and water at a ratio of 80:20, then with pure ethanol and finally dried.
• Hyaff 11 p75 fibres are mixed for at least 10 minutes with 84 cc of a solution constituted by hyaluronic acid in an aqueous solution with a concentration of 18-19 mg/ml.
• Said mixture is supplemented with 20 g. of granules of DM and kneaded for at least 15 minutes.
• the paste thus obtained is inserted in a woven, tubular structure, preferably made of Hyaff 11, and freeze-dried.
• edges are then wetted with a solution of Hyaff 11 in DMSO with a concentration equal to 35 mg/ml, and then immersed in an ethanol bath for at least 10 minutes.
• the product thus obtained is washed in ethanol-water at a ratio of 80:20 for at least 1 hour, then again in pure ethanol and, lastly, dried.

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